MATURATION OF HUMAN PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES PROJECT SUMMARY Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide a novel and physiologically relevant source of cells that have the potential to be used for in vitro cardiotoxicity testing and disease modeling as well as for the study of in vivo heart development and regenerative medicine. However, compared with primary human CMs, hPSC-CMs are structurally and functionally less mature, which limits their ability to accurately predict cardiotoxicity and model human development and diseases, and may result in adverse events such as arrhythmias upon transplantation. Therefore, improving hPSC-CM maturation is paramount in order to improve applications of these cells. We and other groups have previously demonstrated that efficient CM differentiation from hPSCs can be achieved via mimicking molecular signaling of cardiac induction during in vivo cardiogenesis. In our recent publications, we have also shown that microscale tissue engineering enables reliable generation of 3D cardiac spheres that contain highly enriched hPSC-CMs with enhanced sarcomeric structural maturation. We hypothesize that a multipronged, synergistic approach combining tissue engineering with modulation of multiple molecular signaling pathways can efficiently improve hPSC-CM maturation within short culture durations. With this hypothesis, we aim to enable metabolic and functional maturation of hPSC-CMs by providing the cells with (1) appropriate chemical signals to modulate metabolic maturation and (2) suitable environmental cues to mimic the transition from fetal CMs to postnatal CMs. We expect that this study will lead to the establishment of hPSC-CMs with cardiophysiology closer to human hearts, which will facilitate their applications in preclinical and clinical studies, and provide novel insights into molecular regulation of CM maturation.